Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (32).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Pat. 4,603,112, the disclosure of which patent is incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of nonessential DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a nonessential region of pox DNA. The resulting plasmid construct is then amplified by growth within E. coli bacteria (4) and isolated (5,22).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Unperturbed, successful recombination occurs at a frequency of approximately 0.1%.
A basic screening strategy for recovering those viruses modified by a successful recombination involves in situ hybridization of recombinants on replica filters with a radiolabeled probe homologous to the inserted sequences (26,28). A number of modifications have been reported to increase the efficiency of recombination itself or to facilitate the identification of recombinants. Among these modifications are included: using single stranded donor DNA (38); identification of recombinants expressing unique enzymatic functions such as .sup.125 Iododeoxycytidine incorporation into DNA via expression of the Herpes simplex virus thymidine kinase (28); chromogenic substrates for (co)expression of foreign genes along with B galactosidase (3,29); selection for thymidine kinase expression (20,28); antibody based reactions to visualize recombinant plaques (21); use of conditional lethal ts or drug mutants (9,18); selection of recombinants using the neomycin resistance gene from Tn5 and the antibiotic G418 (11); or selection pressures with mycophenolic acid and the E. coli gpt gene (2,8).
Disadvantageously, these known methods for identifying or selecting recombinant poxvirus all involve tedious multi-step identification of the recombinants, the introduction of radiochemicals, chromogenic substrates, biochemicals useful for selection such as mycophenolic acid and bromodeoxyuridine which may be detrimental (mutagenic) to the viral genome itself, use of serological reagents that may introduce contaminants, and typically the presence of an exogenous gene in the final recombinant in addition to the foreign genetic element of interest.
It can thus be appreciated that provision of a method of making and selecting for poxvirus recombinants, particularly vaccinia recombinants, which method avoids the previously discussed problems, would be a highly desirable advance over the current state of technology.